4-bromo-3-hydroxyhippuric acid and cationic salts thereof

ABSTRACT

THIS DISCLOSURE DECRIBES 4-BROMO-3-HYDROXYHIPPURIC ACID AND CATIONIC SALTS THEREOF USEFUL FOR INHIBITING THE FORMATION OF HISTAMINE IN MAMMALS.

U.S. Cl. 260-519 United States Patent O ABSTRACT OF THE DISCLOSURE This disclosure describes 4-bromo-3-hydroxyhippuric acid and cationic salts thereofuseful for inhibiting the formation of histamine in mammals. I

BRIEF SUMMARY OF THE INVENTION 'This invention relates to 4-bromo-3-hydroxyhippuric acid which may be represented by the following structural formula:

I B! Q c-NH- CH2-CO H It is Well established that histamine is implicated in a number of physiological functions including, for example, the nervous system, peripheral circulation, gastric hyperacidity, and allergic manifestations such as hay fever. The decanboxylation of histidine by the specific enzyme histidine decarboxylase results in the formation of histamine in mammals.

Our invention is based upon the discovery that 4-bromo-3-hydroxyhippuric acid is a potent inhibitor of the enzyme histidine decarboxylase with resultant inhibition of histamine formation in mammals. The mode of action of the compounds of the present invention thus differs from that of known antihistaminic compounds which ordinarily merely counteract the existing released or formed histamine.

DETAILED DESCRIPTION OF THE INVENTION Also embraced within the scope of the present invention are the non-toxic, pharmaceutically acceptable cationic salts of 4-bromo-3-hydroxyhippuric acid. Included are the monobasic and dibasic salts of 4-bromo-3-hydroxyhippuric acid. The cations comprised in these salts include, for example, the non-toxic metal cations such as the sodium ion, potassium ion, calcium ion, magnesium ion as well as the organic aminecations such as the tri- (lowenalkyDamine cations (e.g., triethylamine), procaine, and the like.

The effectiveness of the compounds of the present invention in inhibiting the activity of the enzyme histidine decarboxylase was determined by the method of Levine and Watts, Biochemical Pharmacology 15, 841-849 (1966) with the modifications of Ellenbogen et al., Biochemical Pharmacology 18, March 1969. In this procedure, the inhibition of rat gastric histidine dccarboxylase activity was determined by measuring the radioactive carbondioxide released from carboxyl-labeled histidine. The incubation-tubes used in the procedure are described-on page 275 of the text Isotopes in Experimental Pharmacology, L. J. Roth, editor, University of Chicago Press The effectiveness of the compounds of this invention and of brocresine (4-brorno-3-hydroxybenzyloxyamine phosphate, a known inhibitor of histidine decarboxylase) in inhibiting the activity of the rat gastric enzyme histidine dec'arboxylase in vivo was demonstrated by three different procedures as follows;

(A) In vivo inhibition of rat gastric histidine decarboxylase following intraperitoneal doses of 200 milligrams per kilogram of body weight of the indicated compounds was demonstrated. The source of the enzyme was the pyloric portion of the adult rat stomach. The results appear in terms of percent inhibition in Table I below.

TAB LE I 4br0mo-3-hydroxyhippun'c acid, percent Broeresine, percent Timgz) (minutes) after administration:

so::IIIIIIIIIIIIIIIIIIIIIIIIIII (B) In vivo inhibition of gastric free hydrochloric acid secretion in pylorus ligated rats, following the procedure of Shay et al., Gastroenterology 5, 43-61 (1945), was demonstrated. Intraperitoneal doses of the indicated compounds were administered at 200 mg./kg. of body weight. The results appear in terms of percent inhibition of total acid in Table II below.

TABLE II 4-bromo-3-hy- Brocresine, droxyhippuric percent acid, percent Time (minutes) after administration: 35

TABLE III 4-bromo-3-hy- Brocresine, droxyhippuric percent acid, percent Time (minutes) after administration:

4-bromo-3-hydroxyhippuric acid, and the non-toxic pharmaceutically aceptable cationic salts thereof, have thus been found to be highly useful for inhibiting the activity of histidine decarboxylase in mammals when administered in amounts ranging from about 15 mg. to about 200 mg. per kg. of body weight per day. A preferred dosage regimen for optimum results would be from about 15 mg. to about mg. per kg. of body weight per day, and such dosage units are employed that a total of from about 1.0 g. to 7.0 g. of active ingredient for a subject of about 70 kg. body weight are administered in a 24 hour period. The compounds of the present invention may be administered by any convenient route such as orally, intraperitoneally, subcutaneously, intramuscularly or intravenously.

Compositions having the desired clarity, stability, and adaptability for parenteral use are obtained by dissolving from 0.10% to 10.0% by weight of active compound in a vehicle consisting of a polyhydric aliphatic alcohol or mixtures thereof. Especially satisfactory are glycerin, propylene glycol, and the polyethylene glycols. The polyethylene glycols consist of a mixture of non-volatile, normally liquid, polyethylene glycols which are soluble in both water and organic liquids and which have molecular weights of from about 200 to about 1500. Although the amount of active compounddissolved in the above vehicle may vary from 0.10% to 10.0% by weight, it is preferred that the amount of active compound employed be from about 3.0% to about 9.0% by weight. Although various mitxures of the aforementioned nonvolatile polyethylene glycols may be employed, it is preferred to use a mixture having an average molecular weight of from about 200 to about 400.

In addition to the active compounds, the parenteral solutions may also contain various preservatives which may be used to prevent bacterial and fungal contaminattion. The preservatives which may be used for such purpose are, for example, benzyl alcohol, myn'stylgamma-picolinium chloride, phenyl, mercuric nitrate, benzalkonium chloride, phenethyl alcohol, p-chlorophenyl-a-glycerol ether, methyl and propyl parabens, and thimerosal. As a practical matter it is also convenient to employ antioxidants. Suitable antioxidants include, for example, sodium bisulfite, sodium metabisulfite, and sodium formaldehyde sulfoxylate. Generally, from about 0.05% to about 0.2% concentrations of antioxidant are employed.

The preferred concentration of active compound is 25 to 50 mg./ml. of the finished compositions when intramuscular injection is the purpose for which the compositions are intended. They are equally adapted to intravenous administration when diluted with water or diluents employed in intravenous therapy such as isotonic glucose in appropriate quantities. For this use, initial concentrations down to about 10 to 25 mg./ml. of active compound are satisfactory. They are also adapted to oral administration when diluted with drinking water.

The compounds of the present invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the compounds of this invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gum, and the like. Such compositions and preparation should contain at least 0.1% of active compound. The percentage in the compositions and preparations may, of course, be varied and may conveniently be between about to about 75% or more of the weight of the unit. The amount of active compound in such therapeutically useful compositions or preparations is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about and 200 milligrams of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalciurn phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain in addition to materials of the above type a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit, for instance, tablets, pills or capsules may be coated with shellac, sugar, or both. A syrup or elixir may contain the active compounds, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts employed.

The invention will be described in greater detail in conjunction with the following specific examples.

Example 1.-Preparation of 4-bromo-3-hydroxyhippuric acid A 5.0 gram (0.023 mole) portion of 4-bromo3-hydroxybenzoic acid [J.A.C.S. 68, 574 (1946)] is refluxed in 50 ml. of ethanol, containing 1.1 ml, (about 5%) of concentrated sulfuric acid, for three hours. The solution is allowed to cool, 50 ml. of water is added and the mixture is extracted three times with ether. The ether extract is dried over magnesium sulfate for 1.5 hours. The filtered solution is pumped to dryness leaving a white solid which is recrystallized from a 50:50 mixture of ethanol and water. The white crystals are collected by filtration and dried yielding 4.3 grams (75.8%) of ethyl 4-bromo-3-hydroxybenzoate, melting point 83"- 85 C.

The 4.3 grams of ester is refluxed with a 50% molar excess of hydrazine hydrate for 30 minutes yielding a white gummy precipitate. The gum is dissolved in water and cooled causing the formation of 0.92 gram of crystals of the hydrazide derivative.

The 0.92 gram of hydrazide derivative is dissolved in 15 ml. of water containing 0.5 ml. of concentrated hydrochloric acid. The solution is cooled to 0 C. and 5 ml. of an ice cold 10% solution of sodium nitrite is slowly added with stirring. The azide derivative precipitates out, is filtered and washed with cold water.

The azide derivative is suspended in a cold solution of 8 ml. of a 7.5% glycine/water solution. Cold 1 N sodium hydroxide is added dropwise so that the temperature remains below 15 C. When all of the azide has dissolved, the pale yellow solution is warmed to room temperature and acidified to pH 2 with concentrated hydrochloric acid. The solution is extracted with three 10 ml. portions of ethyl acetate and the ethyl acetate is dried over sodium sulfate. The ethyl acetate is removed in vacuo leaving a yellow syrup which upon refrigerating overnight forms crystals (178.34 milligrams) of the desired product, 4 bromo 3 hydroxyhippuric acid, which is recrystallized from ethyl acetate, M.P. 165169 C., yield 170 milligrams.

Example 2.-Preparation of sodium 4-bromo-3- hydroxyhippurate In ml. of methanol was dissolved 27.4 g. of 4- bromo 3 hydroxyhippuric acid and 5.4 g. of sodium methoxide and the resulting solution was evaporated to dryness. There was thus obtained sodium 4 bromo-3- hydroxyhippurate as a white powder.

Example 3.-Preparation of potassium 4-bromo-3- hydroxyhippurate In 100 ml. of water was dissolved 27.4 g. of 4-bromo- 3-hydroxyhippuric acid and 5.6 g. of pellet potassium hydroxide and the resulting solution was evaporated to dryness. There was thus obtained potassium 4- bromo-3- hydroxyhippurate as an 01f white powder.

Example 4.Preparation of tablet formulation The active ingredient, lactose and corn starch (for mix) are blended together. The corn starch (for paste) is suspended in water at a ratio of 10 grams of corn starch per 80 ml. of water and heated with stirring to form a. paste. This paste is then used to granulate the mixed powders. The wet granules are passed through a No. 8 screen and dried at F. The dry granules are passed through a No. 16 screen. The mixture islubricated with magnesium stearate and compressed-into tablets in a suitable tableting machine. Each tablet contains 300 milligrams of active ingredient.

Example 5.Preparation of intramuscular formulation Ingredient: Amount, percent 4-bromo-3-hydroxyhippuric acid 2.5 Parabens (4:1 mixture of methyl and propyl) 0.1 Water for injection 100 The parabens are dissolved in about one-half the volume of Water for injection at 80 C. with stirring. The solution is cooled to below 40 C. and the active ingredient is dissolved therein. The cooled solution is adjusted to final volume with water for injection and is then sterilized by sterile filtration through a suitable filter.

We claim:

1. A compound selected from the group consisting of 4 bromo 3 hydroxyhippuric acid and the non-toxic pharmaceutically acceptable cationic salts thereof.

6 2. 4-bromo-3-hydroxyhippuric acid. 3. Sodium 4-bromo-3-hydroxyhippurate. 4. Potassium 4-bromo-3-hydroxyhippurate.

References Cited UNITED STATES PATENTS 3,489,793 1/1970 Bertelli 260-519 LORRAINE A. WEINBERGER, Primary Examiner L. A. THAXTON, Assistant Examiner US. Cl. X.R. 

